A universal SARS‐CoV DNA vaccine inducing highly cross‐reactive neutralizing antibodies and T cells

Abstract New variants in the SARS‐CoV‐2 pandemic are more contagious (Alpha/Delta), evade neutralizing antibodies (Beta), or both (Omicron). This poses a challenge in vaccine development according to WHO. We designed a more universal SARS‐CoV‐2 DNA vaccine containing receptor‐binding domain loops from the huCoV‐19/WH01, the Alpha, and the Beta variants, combined with the membrane and nucleoproteins. The vaccine induced spike antibodies crossreactive between huCoV‐19/WH01, Beta, and Delta spike proteins that neutralized huCoV‐19/WH01, Beta, Delta, and Omicron virus in vitro. The vaccine primed nucleoprotein‐specific T cells, unlike spike‐specific T cells, recognized Bat‐CoV sequences. The vaccine protected mice carrying the human ACE2 receptor against lethal infection with the SARS‐CoV‐2 Beta variant. Interestingly, priming of cross‐reactive nucleoprotein‐specific T cells alone was 60% protective, verifying observations from humans that T cells protect against lethal disease. This SARS‐CoV vaccine induces a uniquely broad and functional immunity that adds to currently used vaccines.

In their manuscript entitled "Universal SARS-CoV DNA vaccine inducing highly crossrective neutralizing antibodies and T cells" Sofia Appelberg and coworker describe an interesting approach for the development of a more universal Sars-CoV vaccine. Their concept is based on a DNA-vector encoding a fusion protein encompassing the RBD domains of the wt, the beta and alpha variant and the M and N protein. After immunization of mice the authors observe the induction of neutralizing antibodies.
In challenge experiments immunized mice were protected from lethal infection.
There are some open points to be addressed. The authors should show the expression of the OC2.4 construct in transfected cells by immunofluorescence microscopy or Western blot analysis. The analysis of the T cell response is central for the manuscript. The data should be transferred from the supplement into the manuscript The structure of the fusion protein is not fully clear. Is the M protein separated by a P2A sequence from the N protein as described in the text? The titer of S-binding antibodies should be analyzed after the first, second and third vaccination. If the authors have data describing the Th1 /Th2 polarization these data should be included in the supplement Referee #2 (Remarks for Author): Appelberg and colleagues determined the immunogenicity and efficacy of a DNA construct containing receptor binding domain (RBD) loops of the S protein corresponding to the huCoV-19/WH01, Alpha and Beta variants, combined with the M and N proteins of the huCoV-19/WH01 variant to target broadest immune responses. Despite demonstrating some protection against beta virus challenge in mice, there is no evidence that this vaccine is equal or better than licensed vaccines. The main concerns are below: 1. The authors refer to SARS-CoV in the title and text and propose that this approach will give broad reactivity across beta coronaviruses. However, it seems that the vaccine construct contains only genetic elements of SARS-CoV-2, and studies to prove that cross-reactivity or functionality with SARS-CoV or other beta coronaviruses are actually improving the vaccine efficacy are missing. The "SARS-CoV OC-2.4" DNA construct is not clearly described and the author refers to Dai et al Cell 2020 which report a recombinant bivalent RBD approach that does not appear to be relevant for this study. 2. In terms of humoral immunity, the construct does not provide a significant improvement compared to recombinant S protein or spike-DNA. 3. Challenge model with wuhan strain to compare S protein vs DNA is missing, and the challenge model with beta strain does not demonstrate superiority of the DNA construct compared to recombinant S protein. The rationale of use of recombinant S protein + QS21 is not clearly described in the text. 4. T cell cross-reactivity with bat coronavirus is not relevant for the study. Moreover, cross-reactivity levels (figure S3) are below threshold of detection for 4 mice out of 6. 5. Evidence of 60% protection with N protein immunization is not supported histological and RNA evidence, with worsening of clinical score in animals post challenge compared to other treatments. Moreover, this is not relevant for the DNA vaccine. 6. Statistical values, experimental replicates and description of experimental conditions are often missing in the figures and text. 7. There are plenty of typos in the title, abstract, text and figures that makes it difficult to understand the manuscript.
Overall, the manuscript is difficult to read and the conclusions are not supported by relevant experimental evidence. Despite the aim of expanding breadth of response, additional work is necessary to clarify rationale of the study, to improve the DNA construct and to generate the right controls and the missing data. Thus, I believe the manuscript is not suitable for publication.

Referee #3 (Remarks for Author):
In this elegant study, the group of Matti Sallberg from Sweden describes a universal SARS-CoV DNA vaccine that induces highly cross-reactive neutralizing antibodies and T cells. This group is leading in vaccine development and the data shown convincing. The transgenic moused model used supports the protective role of the induced immune response and also supports a critical role of T cells as has been suggested in the hunan system. Overall, in this reviewers opinion, this is a well performed study that addresses an important and highly relevant topic.
I have only one comment: The authors use different vaccine strategies, the rationale for which should be discussed or at least be explained.
In their manuscript entitled "Universal SARS-CoV DNA vaccine inducing highly crossrective neutralizing antibodies and T cells" Sofia Appelberg and coworker describe an interesting approach for the development of a more universal Sars-CoV vaccine. Their concept is based on a DNA-vector encoding a fusion protein encompassing the RBD domains of the wt, the beta and alpha variant and the M and N protein. *If the authors have data describing the Th1 /Th2 polarization these data should be included in the supplement REPLY: We have focused on the cross reactivity of the T cells, also the strong IFNg responses certainly suggest a Th1-like response. This will however be included in the next publication.

Referee #2 (Remarks for Author):
Appelberg and colleagues determined the immunogenicity and efficacy of a DNA construct containing receptor binding domain (RBD) loops of the S protein corresponding to the huCoV-19/WH01, Alpha and Beta variants, combined with the M and N proteins of the huCoV-19/WH01 variant to target broadest immune responses. Despite demonstrating some protection against beta virus challenge in mice, there is no evidence that this vaccine is equal or better than licensed vaccines. The main concerns are below: 1. The authors refer to SARS-CoV in the title and text and propose that this approach will give broad reactivity across beta coronaviruses. However, it seems that the vaccine construct contains only genetic elements of SARS-CoV-2, and studies to prove that cross-reactivity or functionality with SARS-CoV or other beta coronaviruses are actually improving the vaccine efficacy are missing. The "SARS-CoV OC-2.4" DNA construct is not clearly described and the author refers to Dai et al Cell 2020 which report a recombinant bivalent RBD approach that does not appear to be relevant for this study.
REPLY: Thank you for the comment. The description of the SARS-CoV OC-2.4 vaccine has now been better described. We agree that much of the data provided show cross reactivity between SARS-CoV-2 variants. However, we clearly show that vaccine primed T cells are cross reactive with Bat-SARS-CoV. Hence, we feel that it is correct to state that the OC-2.4 vaccine induces immune responses cross reactive with SARS-CoVs. *2. In terms of humoral immunity, the construct does not provide a significant improvement compared to recombinant S protein or spike-DNA.
REPLY: This is correct. However, the major point of the current vaccine design is to activate both humoral and broadly reactive T cells. Thus, we feel that it is quite impressive that the anti-S responses are comparable to S-based vaccines, but with the addition that new responses to M and N are induced.
*3. Challenge model with wuhan strain to compare S protein vs DNA is missing, and the challenge model with beta strain does not demonstrate superiority of the DNA construct compared to recombinant S protein. The rationale of use of recombinant S protein + QS21 is not clearly described in the text.
REPLY: We feel that the most relevant control is the Beta variant. Also, we agree that the vaccine does not show superiority in the in vivo model. However, it is important to stress that we do induce new T cell specificities to highly conserved parts of the virus, such as M and N. This suggests, together with the T cell data, that we have a vaccine that complements the existing vaccines by adding new T cell repertoires. Importantly, as we show, the activation of N-specific T cells alone has protective properties, supporting the concept.
*4. T cell cross-reactivity with bat coronavirus is not relevant for the study. Moreover, cross-reactivity levels (figure S3) are below threshold of detection for 4 mice out of 6.
REPLY: We disagree with the reviewer's comment. The cross reactivity with Bat N sequences is central to a universal SARS-CoV vaccine, since new viruses are likely to appear from animals such as bats. Thus, even if the S gene is replaced, a prior SARS-CoV infection, or vaccination using our type of vaccine, will still have the chance of offering some degree of protection. Indeed, that is what the experiment with vaccination with N protein alone shows. Thus, the cross reactivity of the vaccineprimed T cells to Bat N sequences is highly relevant.
*5. Evidence of 60% protection with N protein immunization is not supported histological and RNA evidence, with worsening of clinical score in animals post challenge compared to other treatments. Moreover, this is not relevant for the DNA vaccine.
REPLY: We disagree with the reviewer as we feel that the data showing that priming T cells only has a partially protective effect is highly relevant. This is a novel observation that extends data from humans regarding the role of T cells. Although there is histological signs of disease the increased survival clearly shows benefit of priming N specific T cells. Thus, although this was a protein-based vaccination it supports the concept of a protective role of cross-reactive T cells. Importantly, the N protein was from the WH1 strain and protected against a challenge with a Beta strain. Overall, the manuscript is difficult to read and the conclusions are not supported by relevant experimental evidence. Despite the aim of expanding breadth of response, additional work is necessary to clarify rationale of the study, to improve the DNA construct and to generate the right controls and the missing data. Thus, I believe the manuscript is not suitable for publication.
Referee #3 (Remarks for Author): In this elegant study, the group of Matti Sallberg from Sweden describes a universal SARS-CoV DNA vaccine that induces highly cross-reactive neutralizing antibodies and T cells. This group is leading in vaccine development and the data shown convincing. The transgenic moused model used supports the protective role of the induced immune response and also supports a critical role of T cells as has been suggested in the human system. Overall, in this reviewers opinion, this is a well performed study that addresses an important and highly relevant topic.
I have only one comment: The authors use different vaccine strategies, the rationale for which should be discussed or at least be explained.
REPLY: This has now been explained in the text. Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. We have now heard back from the two referees who we asked to re-evaluate your manuscript. As you will see from the reports below, while the referee #1 supports publication of your manuscript, referee #2 acknowledges the improvements of the revised manuscript but also raises a number of concerns that should be addressed in an additional and final round of major revision. Only one point considering a control group immunized with N protein + QS21 requires additional experimentation, while all the other points should be addressed by additional clarifications and discussion.
Further consideration of a revision that addresses reviewer's concerns in full will entail an additional round of review. Acceptance or rejection of the manuscript will depend on the completeness of your responses included in the next, final version of the manuscript. For this reason, and to save you from any frustrations in the end, I would strongly advise against returning an incomplete revision.
We would welcome the submission of a revised version within three months for further consideration. Please let us know if you require longer to complete the revision.
Please use this link to login to the manuscript system and submit your revision: Link Not Available The authors adequately addressed all points raised in the previous review.
Referee #1 (Remarks for Author): The authors adequately addressed all points raised in the previous review. This is a very relevant, well designed and well performed study.

Referee #2 (Comments on Novelty/Model System for Author):
Please see suggested experiments and manuscript review in the comments to the authors.

Referee #2 (Remarks for Author):
The authors focus their discussion on the benefit of inducing N specific T cell response which are cross-reactive with other coronaviruses. When recombinant N protein + adjuvant is used, it results in 60% protection against challenge ( Figure 3C). The DNA vaccine is reported to induce both N and spike immunity (Figure 2B,F,G), broadening the immune response, however no added protection is observed in the challenge model (recombinant Wh01 spike protein and DNA vaccine confer full protection against beta challenge, Figure 3C). The study has several limitations which the authors have highlighted in the new version and the flow is clearer. However, I believe the significance of this is limited, and it could be considered for publication provided additional comments are addressed.
Additional comments: • Clarify why QS21 adjuvant was used • Remove statements related N specific responses whose data is not shown • Regarding Figure 2C, additional M and N specific T cell are induced after booster however no significance analysis is given, this should be stated in the text.
• Figure 2E-H should be clearly described in the results section, clarifying that specific IFNy responses are below limit of detection for several mice/group. • A control group in Figure 2 immunized with N protein + QS21 is missing, this is necessary to define the level of N specific immunity that correlates with in vivo protection, and to compare with DNA-induced immunity • Clarify in figure le legend and materials&methods what is 1-10 in x axis of Figure 2A Replies to comments made by the reviewers.

Referee #1 (Comments on Novelty/Model System for Author):
The authors adequately addressed all points raised in the previous review.
Referee #1 (Remarks for Author): The authors adequately addressed all points raised in the previous review. This is a very relevant, well designed and well performed study.
We thank the reviewer for the comment.

Referee #2 (Comments on Novelty/Model System for Author):
Please see suggested experiments and manuscript review in the comments to the authors.

Referee #2 (Remarks for Author):
The authors focus their discussion on the benefit of inducing N specific T cell response which are cross-reactive with other coronaviruses. When recombinant N protein + adjuvant is used, it results in 60% protection against challenge ( Figure 3C). The DNA vaccine is reported to induce both N and spike immunity (Figure 2B,F,G), broadening the immune response, however no added protection is observed in the challenge model (recombinant Wh01 spike protein and DNA vaccine confer full protection against beta challenge, Figure 3C). The study has several limitations which the authors have highlighted in the new version and the flow is clearer. However, I believe the significance of this is limited, and it could be considered for publication provided additional comments are addressed.
Additional comments: • Clarify why QS21 adjuvant was used REPLY: The use of the QS21 adjuvant was based on the fact that it is commercially available for research and clinical use. Also, new Expanded View Figure 3 shows that QS21 is superior to alumn, the today most widely used vaccine adjuvant in humans.
• Remove statements related N specific responses whose data is not shown REPLY: We have now included data that support the statement regarding N antibodies on line 10, page 5. 7th Jul 2022 2nd Authors' Response to Reviewers • Regarding Figure 2C, additional M and N specific T cell are induced after booster however no significance analysis is given, this should be stated in the text.
REPLY: This has now been included in the text. Also, statistical comparisons have been included for both mice and rabbit T cell responses.
• Figure 2E-H should be clearly described in the results section, clarifying that specific IFNy responses are below limit of detection for several mice/group.
REPLY: This has now been clarified in the text that some rabbits failed to develop any T cell responses at any time point.
• A control group in Figure 2 immunized with N protein + QS21 is missing, this is necessary to define the level of N specific immunity that correlates with in vivo protection, and to compare with DNA-induced immunity REPLY: An additional experiment has been added to clarify this issue, Extended View 3. Also described in Results on page 10.
• Clarify in figure le legend and materials&methods what is 1-10 in x axis of Figure  2A-D REPLY: Thank you for noticing this. This has now been added in the legend, that this corresponds to peptide pools.
• Add p values asterisks in figure 3D REPLY: This has now been done. -Data availability statement should contain information about data that cannot be published in the manuscript itself (e.g. structural data, high-throughput sequencing or data from large-scale gene expression experiments). If no data are deposited in public repositories, please add the sentence: "This study includes no data deposited in external repositories".
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I look forward to reading a new revised version of your manuscript as soon as possible.

Zeljko Durdevic
Zeljko Durdevic Editor EMBO Molecular Medicine ***** Reviewer's comments ***** Referee #2 (Remarks for Author): The authors addressed most of the concerns on the manuscript adding supplementary experimental data and adding more details in the main text. I am ok for publication.